Albumin is the most abundant plasma protein, generally constituting slightly over one-half of the total protein in mammalian plasma. In the human body, albumin has the important role of regulating the water balance between blood and tissues, and of functioning as a transport molecule for various compounds, such as bilirubin, fatty acids, cortisol, thyroxine and drugs such as sulfonamides and barbiturates, that are only slightly soluble in water. An albumin deficiency can restrict the transport of slightly water soluble materials throughout the body and a deficiency is signaled in an individual by an abnormal accumulation of serous fluid, or edema. Therefore, it is clinically important to determine whether an individual has a deficiency of serum albumin.
Likewise, it is clinically important to determine if an individual is excreting an excess amount of protein. A normal functioning kidney forms urine in an essentially two step process. Blood flows through the glomerulus, or glomerular region of the kidney. The capillary walls of the glomerulus are highly permeable to water and low molecular weight components of the blood plasma. Albumin and other high molecular weight proteins cannot pass through these capillary walls and are essentially filtered out of the urine so that the protein is available for use by the body. The liquid containing the low molecular weight components passes into the tubules, or tubular region, of the kidney where reabsorption of some urine components, such as low molecular weight proteins; secretion of other urine components; and concentration of the urine occurs. As a result, through the combined processes of the glomerulus and tubules, the concentration of proteins in urine should be minimal to non-existent Therefore, abnormally high amounts of albumin and/or low-molecular weight proteins in urine must be detected and related to a physiological dysfunction.
The relatively high concentration of albumin in the urine of an individual usually is indicative of a diseased condition. For example, the average normal concentration of protein in urine varies from about 2 mg/dL to about 8 mg/dL, with approximately one-third of the total urinary protein being serum albumin. However, in a majority of diseased states, urinary protein levels increase appreciably, such that albumin accounts for from about 60 percent to about 90 percent of the excreted protein. The presence of an abnormal increased amount of protein in the urine, known as proteinuria, is one of the most significant indicators of renal disease, and may be indicative of various other non-renal related diseases.
Therefore, in order to determine if an individual has an albumin deficiency and/or to determine if an individual excretes an excess amount of protein, and in order to monitor the course of medical treatment to determine the effectiveness of the treatment, simple, accurate and inexpensive protein detection assays have been developed. Furthermore, of the several different assay methods developed for the detection and/or measurement of protein in urine and serum, the methods based on dye binding techniques have proven especially useful because dye binding methods are readily automated and provide reproducible and accurate results.
In general, dye binding techniques utilize pH indicator dyes that are capable of interacting with a protein, such as albumin, and that are capable of changing color upon interaction with a protein absent any change in pH. When a pH indicator dye interacts with, or binds to, a protein, the apparent pK.sub.a (acid dissociation constant) of the indicator dye is altered and the dye undergoes a color transition, producing the so-called "protein-error" phenomenon. In methods utilizing the dye binding technique, an appropriate buffer maintains the pH indicator dye at a constant pH to prevent a color transition of the pH indicator dye due to a substantial shift in pH. Due to the "protein-error" phenomena, upon interaction with the protein, the pH indicator dye undergoes a color transition that is identical to the color change arising because of a change in the pH. Examples of pH indicator dyes used in the dry phase assay of proteins that are capable of interacting with or binding to proteins and exhibiting "protein-error" color transitions include tetrabromophenol blue and tetrachlorophenol-3,4,5,6-tetrabromosulfophthalein.
Although pH indicator dyes have been used extensively in protein assays, several problems and disadvantages still exist in protein assay methods utilizing indicator dyes. For example, methods based upon pH indicator dyes either cannot detect or cannot quantitatively differentiate between protein concentrations below approximately 15 mg/dL. In addition, although several simple semiquantitative tests and several complex quantitative tests are available for the determination of the total protein content in a test sample, the majority of these assay methods, with the notable exception of the simple colorimetric reagent test strip, require the precipitation of protein to make quantitative protein determinations.
The colorimetric reagent test strip utilizes the previously discussed ability of proteins to interact with certain acid-base indicators and to alter the color of the indicator without any change in the pH. For example, when the indicator tetrabromophenol blue is buffered to maintain a constant pH of approximately 3, the indicator imparts a yellow color to solutions that do not contain protein. However, for solutions containing protein, the presence of protein causes the buffered dye to impart either a green color or a blue color to the solution, depending upon the concentration of protein in the solution.
Some colorimetric test strips used in protein assays have a single test area consisting of a small square pad of a carrier matrix impregnated with a buffered pH indicator dye, such as tetrabromophenol blue. Other colorimetric test strips are multideterminant reagent strips that include one test area for protein assay as described above, and further include several additional test areas on the same strip to permit the simultaneous assay of other urinary constituents. For both types of colorimetric test strips, the assay for protein in urine is performed simply by dipping the colorimetric test strip into a well mixed, uncentrifuged urine sample, then comparing the resulting color of the test area of the test strip to a standardized color chart provided on the colorimetric test strip bottle.
For test strips utilizing tetrabromophenol blue, buffered at pH 3, as the indicator dye, semiquantitative assays for protein can be performed and are reported as negative, trace, or one "plus" to four "plus". A negative reading, or yellow color, indicates that the urine contains no protein, as demonstrated by the lack of a color transition of the indicator dye. A trace reading may indicate from about 5 to about 20 mg/dL of protein in the urine. The one "plus" to four "plus" readings, signified by color transitions of green through increasingly dark shades of blue, are approximately equivalent to urine protein concentrations of 30, 100, 300, and over 2000 mg/dL, respectively, and serve as reliable indicators of increasingly severe proteinuria.
In accordance with the above-described method, an individual can readily determine, visually, that the protein content of a urine sample is in the range of 0 mg/dL to about 30 mg/dL However, the color differentiation afforded by the presently available commercial test strips is insufficient to allow an accurate determination of protein content in urine between 0 mg/dL and about 15 mg/dL. The inability to detect and differentiate between low protein concentrations is important clinically because a healthy patient usually has a urine protein level in the range of about 10 mg/dL to about 20 mg/dL. Therefore, it could be clinically important to know more precisely the urine protein content of an individual, rather than merely estimating the protein content at some value less than about 30 mg/dL.
Of course, the protein content of a urine sample can be determined more precisely by semiquantitative protein precipitation techniques or by quantitative 24 hour protein precipitation techniques. However, these tests are time consuming and relatively expensive. Furthermore, the precipitation tests must be run in a laboratory by trained personnel, and therefore are unavailable for the patient to perform at home to quickly determine urine protein content and to monitor the success or failure of a particular medical treatment
Therefore, it would be extremely advantageous to have a simple, accurate and trustworthy method of assaying urine for a low to trace protein content that allows visual differentiation of protein levels in the ranges of 0 mg/dL to about 5 mg/dL, about 5 mg/dL to about 10 mg/dL, about 10 mg/dL to about 20 mg/dL, and about 20 mg/dL to about 30 mg/dL, and upwards to between about 100 mg/dL to about 300 mg/dL. By providing such an accurate method of determining a low to trace urine protein concentration in an easy to use form, such as a dip-and-read test strip, the urine assay for low to trace amounts of protein can be performed by laboratory personnel to afford immediate test results such that a diagnosis can be made without having to wait up to one day for assay results, and medical treatment can be commenced immediately. In addition, the test strip method can be performed by the patient at home to more precisely monitor low to trace levels of protein in urine and/or the success of the medical treatment the patient is undergoing.
As will be described more fully hereinafter, the method of the present invention allows the fast, accurate and trustworthy assay for low to trace levels of protein in urine by utilizing a test strip that includes a reagent composition incorporating a tungstate-dye complex. The tungstate-dye complex reagent composition improves the sensitivity of the assay and provides sufficient visual color resolution between different protein concentrations, and therefore allows urine protein concentrations at levels of approximately 30 mg/dL or less to be accurately determined. In addition, the method of the present invention also can be used to determine the presence and/or concentration of higher concentrations of proteins, such as from about 100 mg/dL to about 2000 mg/dL, in a test sample.
The level of proteinuria exhibited by an individual depends upon the precise nature of the clinical and pathological disorder and upon the severity of the specific disease. Proteinuria can be intermittent or continuous, with transient, intermittent proteinuria usually being caused by physiologic or functional conditions rather than by renal disorders. Therefore, accurate and thorough assays of urine and other liquid test samples for protein must be available for both laboratory and home use. The assays must permit the detection and measurement even of low to trace concentrations of proteins, such that a correct diagnosis can be made and correct medical treatment implemented, monitored and maintained. In addition, it would be advantageous if the protein assay method for low to trace concentrations of protein could be utilized in a dip-and-read format for the easy and economical, qualitative and/or semiquantitative determination of protein in urine or other liquid test samples.
Furthermore, any method of assaying for protein in urine or other test samples must yield accurate, trustworthy and reproducible results by utilizing a reagent composition that undergoes a color transition as a result of an interaction with protein, and not as a result of a competing chemical or physical interaction, such as a pH change or preferential interaction with a test sample component other than protein. Moreover, it would be advantageous if the protein assay method is suitable for use both in wet assays and in dry reagent strips for the rapid, economical and accurate determination of protein in urine or other test samples. Additionally, the method and composition utilized in the assay for protein should not adversely affect or interfere with the other test reagent pads that are present on multiple test pad strips.
Prior to the present invention, no known method of assaying urine or other test samples for proteins included a reagent composition that provides sufficient sensitivity and color resolution of the assay at low to trace protein concentration levels, such that accurate and trustworthy protein assays can be made for protein concentrations of about 30 mg/dL and below. In addition, although a dry phase chemistry test strip utilizing a single dye, such as tetrabromophenol blue or tetrachlorophenol-3,4,5,6-tetrabromosulfophthalein, has been used extensively for several years, no dry phase test strip has incorporated a tungstate-dye complex to provide sufficient sensitivity, and therefore sufficient visual color-resolution between protein levels at low to trace protein concentration levels.
The prior art contains numerous references on the wet phase and the dry phase chemistry utilized in the pH indicator dye method of assaying urine for proteins. For example, Keston U.S. Pat. No. 3,485,587 discloses the basic dye binding technique used to assay for proteins at a constant pH. Keston teaches utilizing a single indicator dye, maintained at a constant pH slightly below the pK.sub.a (acid dissociation constant) of the dye, to determine the presence and/or concentration of albumin by monitoring the color transition of the dye.
The publication "Color Reaction Between Pyrogallol Red-Molybdenum (VI) Complex and Protein", Y. Fujita, I. Mori, and S. Kitano, Bunseki Kaqaku, 32, pp. E379-E386 (1983), describes the protein interaction with a pyrogallol red-molybdenum complex requiring incorporation of a chelating agent or metal ion into the complex in order to determine protein concentrations.
Similarly, Japanese Patent No. 61/155757 (1986) discloses a colorimetric method of assaying for trace amounts of proteins in a test sample by using a composition including a molybdenum-dye complex and either a chelating agent or certain metal ions. However, it has been found that the method disclosed in Japanese Patent No. 61/155757 suffers from a severe ionic strength and specific gravity interference such that the extent of molybdate-dye binding to the protein, and therefore the degree of color transition, is inversely related to the ionic strength of the sample. Therefore, the assay of a urine sample of low ionic strength (low specific gravity) produces a greater color transition in the test device (therefore indicating a greater protein content) than the assay of a urine sample having the same protein content, but a higher ionic strength (higher specific gravity). Unexpectedly, the tungstate-dye complex utilized in the present invention does not suffer from an ionic strength/specific gravity interference and provides accurate protein assays regardless of test sample ionic strength Furthermore, it has been found that the inclusion of a chelating agent, added to the composition disclosed in the Japanese patent to suppress the blank reaction, is unnecessary and actually is detrimental to the method of the present invention.
The method disclosed in Japanese Patent No. 61/155757 also was described in the publication, "Urinary Protein as Measured with a Pyrogallol Red-Molybdate Complex, Manually and in a Hitachi 726 Automated Analyzer", N. Watanabe, S. Kamei, a. Ohkubo, M. Yamanaka, S. Ohsawa, K. Makino and K. Tokuda, Clin. Chem., 32/8, pages 1551-1554 (1986). This publication describes the automated or manual detection of proteins in urine using the molybdate-dye complex. In addition to the disadvantages described above, the publication further reports that the interaction of interest between the protein and the molybdate-dye complex continued for at least eight minutes and is complete within 10 minutes at 37.degree. C. for automated assays, but for manual assays, the interaction was allowed to continue for 20 minutes before the assay was examined for a response. Such a long interaction-time for the complete color transition to occur is both inconvenient and can lead to erroneous assays should the degree of color transition, and hence protein content, be determined too quickly. However, according to the method of the present invention, the interaction between the protein and the tungstate-dye complex is complete in less than two minutes, therefore providing faster results with a greatly reduced probability of an erroneous assay.
In contrast to the prior art, and in contrast to the presently available commercial test strips, the method of the present invention provides increased sensitivity in the detection and measurement of proteins in urine, especially low to trace amounts of proteins, by utilizing a reagent composition including a tungstate-dye complex, such that accurate assay for protein levels of about 30 mg/dL and below is achieved. Unexpectedly and surprisingly, the method of the present invention, also in contrast to the prior art, also allows the simple and fast detection and measurement of low to trace levels of protein in a liquid test sample. Hence, in accordance with the method of the present invention, new and unexpected results are achieved in the dry phase reagent strip assay and in the wet assay of urine and other test samples for proteins, especially for low to trace concentrations of proteins, by utilizing a reagent composition including a tungstate-dye complex.